Molecular marker primer of white gourd fusarium wilt resistance major gene fob2 and marking method and application thereof

Abstract

The invention relates to the technical field of molecular genetics, in particular to a molecular marker primer of a white gourd fusarium wilt resistant major gene fob2 and a marking method and application of the molecular marker primer. According to the molecular marker primer, a high white gourd fusarium wilt resistant material HN1-1 and a high fusarium wilt susceptible material 159 are hybridized to obtain F1 heterozygous species, then selfing is conducted on the F1 heterozygous species to obtain an F2 genetic population, and the F2 genetic population is subjected to hybridization to obtain the white gourd fusarium wilt resistant major gene fob2. The genetic linkage analysis is carried out through the genotype of each single plant of the F2 group and the phenotype of the disease resistance level of fusarium wilt to identify that the white gourd material HN1-1 carries a recessive disease-resistant major gene, and the recessive disease-resistant major gene is named fob2. Molecular markers 9M42.56 and 9M46.89 are developed according to the major gene, the molecular markers can effectively detect whether a white gourd disease-resistant material HN1-1 and derivative strains or varieties thereof carry the major resistance gene or not, and the selection efficiency of positive plants in white gourd fusarium wilt resistance breeding is greatly improved.

Description

[Technical field]

[0001] The invention relates to the technical field of molecular genetics, and in particular to a molecular genetics primer of a wax gourd wilt resistance major gene fob2 and a labeling method and application thereof. [Background technology]

[0002] Winter melon (Benincasa hispida Cogn.) is an annual vine herbaceous plant of the genus Wax melon in the Cucurbitaceae family. It has high nutritional value and therapeutic effects. It is an important vegetable crop and food processing raw material for my country's southern vegetables to the north. At present, my country's winter melon cultivation is mainly concentrated in Guangxi, Guangdong, Hainan and other regions in South China. With the large-scale and regionalized concentrated planting of a single variety of winter melon for many years, the frequent occurrence of wilt has seriously damaged winter melon production. The wilt damage has become a bottleneck restricting the development of my country's winter melon industry. In addition to conventional field prevention and control, breeding winter melon varieties or strains with high resistance to wilt is also the most economical, effective, green and safe measure to prevent and control wilt damage. There are few varieties of winter melon with high resistance to wilt and it is difficult to breed and utilize them. It requires multi-parent hybridization, backcrossing and self-pollination breeding, and the operation is cumbersome and complicated. In addition, the genetics of winter melon resistance to wilt are complex and difficult to utilize. The resistance of wax gourd to Fusarium wilt is controlled by multi-gene recessive inheritance, which conforms to the "additive-dominant" model, in which the additive effect is dominant and the susceptibility to disease resistance is partially dominant.

[0003] Due to the tediousness and complexity of disease resistance identification, the use of molecular assisted selection (MAS) technology can effectively solve this problem and improve the efficiency of breeding. However, there are few reports on the use of MAS for breeding of wilt resistance. Before this application, the research team developed three molecular markers (3M9.425, 3M13.385 and 3M16.869) closely linked to the Fob1 resistance site for chromosome 3, but no research has been conducted on chromosome 9 of this application.

[0004] Therefore, based on the detection of a recessive major gene fob2 for resistance to wilt disease on chromosome 9 of wax gourd and the development of molecular markers that are closely linked and co-segregated with the gene, this application conducts in-depth design and modification of several pairs of molecular markers to obtain two pairs of simple and easy-to-use functional markers 9M42.56 and 9M46.89. With the help of MAS technology, disease-resistant genes can be effectively introduced, disease-resistant positive plants or lines or varieties can be selected, and the breeding period of wax gourd resistant to wilt disease varieties can be shortened. [Summary of the invention]

[0005] In view of the above, it is necessary to provide a molecular marker primer for the major gene fob2 of wax gourd resistance to Fusarium wilt and its marking method and application. This molecular marker can provide new ideas for future research on genetic breeding, genome mapping, gene positioning and species relationship identification.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The invention discloses an application of a primer set for detecting molecular markers of fob2, a major effect gene for resistance to wilt disease in wax gourd, in breeding wax gourd strains or varieties with high resistance to wilt disease. The primer set comprises primer pair 9M42.56 and / or primer pair 9M46.89; the upstream primer sequence of the primer pair 9M42.56 is: 5'-CCTTATTTAACCGATGTGAT-3'; the downstream primer sequence is: 5'-AGATTGGGAAG TCTTAGAGC-3' (SEQ ID NO.1-2); the upstream primer sequence of the primer pair 9M46.89 is: 5'-GGATCTC CTACATGGTGCCT-3'; the downstream primer sequence is: 5'-TAATGCTTTAGGCTGAAGAG-3' (SEQ ID NO.3-4); and the wax gourd material is HN1-1.

[0008] The present invention also includes a method for breeding wax gourd strains or varieties with high resistance to Fusarium wilt using the primer set, the method comprising:

[0009] Primer pair 9M42.56 is used to amplify the genomic DNA of the wax gourd sample to be tested. If a 294 bp target fragment can be amplified, it means that the wax gourd sample contains the major gene fob2 for resistance to wax gourd wilt disease. If a 294 bp target fragment cannot be amplified, it means that the wax gourd sample does not contain the major gene fob2 for resistance to wax gourd wilt disease.

[0010] Or use primer pair 9M46.89 to amplify the genomic DNA of the wax gourd sample to be tested. If the target fragment of 159 bp can be amplified, it means that the wax gourd sample contains the major gene fob2 for resistance to wax gourd wilt disease; if the target fragment of 159 bp cannot be amplified, it means that the wax gourd sample does not contain the major gene fob2 for resistance to wax gourd wilt disease.

[0011] The present invention has the following beneficial effects:

[0012] 1. The present invention identified a recessive major gene for resistance to wax gourd wilt disease for the first time using molecular markers and named it fob2.

[0013] 2. The main effect gene position of the molecular marker detection developed by the present invention is clear and easy to identify. By detecting the molecular markers closely linked to the gene, it is possible to accurately determine whether the intermediate breeding material carries wilt resistance, and then quickly screen disease-resistant strains or varieties. The detection is convenient, fast and easy, and is not affected by the environment.

[0014] 3. The auxiliary breeding selection has clear goals and saves costs. In the traditional breeding method, it is first necessary to collect disease-resistant materials, create intermediate materials through a series of ways such as hybridization, backcrossing and self-pollination, and carry out wilt resistance identification and selection. The operation is cumbersome and complicated and easily affected by the environment. In addition, before the disease resistance identification, it is necessary to separate and obtain a specific bacterial source and cultivate and propagate the pathogenic bacteria population. It is also necessary to control the concentration of the inoculated bacterial source and synchronize the seedling age of the melon seedlings to be tested. The requirements are harsh and challenging. If the relationship between the bacterial source, melon seedlings and the environment is not well coordinated, the reliability of the phenotypic identification of wilt resistance is directly reduced, affecting the accuracy of the experimental results. Therefore, the traditional winter melon disease resistance breeding cycle is long, difficult and costly; and the method of the present invention for detecting the main effect gene loci of disease resistance through molecular markers is used for the genotype detection of strains or varieties in winter melon breeding. Positive single plants carrying resistance genes can be identified at the winter melon seedling stage, and other plants that do not carry resistance genes are eliminated, which greatly improves the screening efficiency of winter melon resistance to wilt, saves production costs, and promotes the breeding process of winter melon disease-resistant strains or varieties.

Brief Description of the Drawings

[0015] Figure 1 Donggua 159, HN1-1 and F 1 Phenotypic identification of plant resistance to Fusarium wilt.

[0016] Figure 2 Donggua159 / HN1-1 F 2 Phenotypic distribution of population resistance to Fusarium wilt.

[0017] Figure 3 This is the location map of the major gene fob2 for resistance to Fusarium wilt in wax gourd material HN1-1 on chromosome 9.

[0018] Figure 4 This is the agarose gel electrophoresis detection diagram of the amplified products of molecular marker 9M42.56 in different wax gourd materials.

[0019] Figure 5 This is the agarose gel electrophoresis detection diagram of the amplified products of molecular marker 9M46.89 in different wax gourd materials. Figure 4-Figure 5 In the figure, M is BM2000 DNA marker, and the band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, respectively; R is HN1-1, S is 159, and lanes 1-10 are part of 159 / HN1-1 F2:3 Family, lanes 11-14 are 4 negative wax gourd materials that do not carry the fob2 gene; the arrows point to the target band of the resistant parent. In the positive samples, there are target bands (294bp, 9M42.56, Figure 4 ; or 159bp, 9M46.89, Figure 5 ), while this band was not present in negative samples. [Specific implementation method]

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0021] The parents used in the examples of the present application: Highly resistant HN1-1 was collected by the applicant from the wild in Nanning, Guangxi Zhuang Autonomous Region, China, and Highly sensitive 159 was collected by the applicant from the wild in Yulin, Guangxi Zhuang Autonomous Region, China; they are now stored in the germplasm resource bank of our institute, and the applicant promises that any unit or individual can obtain the above varieties from our unit.

[0022] Example 1

[0023] This embodiment is a scheme for obtaining molecular markers, which is as follows:

[0024] 1. Construction of six-generation materials and phenotypic identification

[0025] (1) Sixth Generation (P 1 , P 2 、F 1 、F 2 , B.C. 1 P 1 , B.C. 1 P 2 The previous disease resistance identification experiment showed that the wax gourd material HN1-1 has high resistance to the oxysporum Schl. f. sp. benincasae bacteria. In order to locate and clone the resistance site, the present invention used the wax gourd highly susceptible material 159 as the female parent and the highly resistant material HN1-1 as the male parent, and hybridized to obtain F 1 , F 1 BC was obtained by backcrossing with both parents 1 P 1 and B.C. I P 2 , F1 159 / HN1-1 F was obtained by self-pollination 2 Genetic segregation population, six generations of materials used for genetic law and phenotypic identification of resistance to Fusarium wilt in wax gourd; F 2 The corresponding F 2:3 Family, used for screening of recombinant individual strains.

[0026] (2) The root system bacterial solution immersion method was used to identify the resistance of winter melon to wilt disease. The strain was the Fusarium oxysporum Schl. f. sp. Benincasae, which was isolated, identified and preserved by the project team. The culture medium was made of corn flour: water: rice bran in a ratio of 2:5:1. After inoculation, it was cultured in an incubator at 30°C for 6 days. After the pathogen produced a large number of spores, it was prepared into a concentration of 5×10 5 / mL of spores of wax gourd wilt pathogen; the sixth-generation wax gourd seedlings at the 2-leaf 1-heart stage were inoculated using the root system bacterial solution immersion method, and the roots were transplanted to cultivation bags (h=20cm, d=20cm) for planting after the roots were soaked in the bacterial solution for 5 minutes, and conventional production management was carried out; the disease situation of the plants was observed every day, and records were made when the highly susceptible material 159 began to develop the disease, and records were made once every 2 days until the disease resistance score (DRS) of 159 was close to level 4, and the disease level index of each material was registered. The disease resistance value standard is divided into 6 resistance levels, with levels 0 and 1 representing high resistance levels, levels 2 and 3 representing intermediate types, and levels 4 and 5 representing high sensitivity levels. Level 0 means healthy plant growth with no leaf damage; level 1 means one true leaf wilts; level 2 means two true leaves wilt; level 3 means three true leaves wilt; level 4 means four true leaves wilt; level 5 means all cotyledons wilt and die. The resistance level of each strain is the weighted average of the measured levels of each repeated strain.

[0027] 2. 159 / HN1-1 F 2 Molecular marker analysis of populations

[0028] (1) According to F 2 The average disease resistance level of the population was determined by selecting 30 extremely resistant plants and 30 extremely susceptible plants, extracting their leaf genomic DNA using the CTAB method (Murray et al. 1980), mixing them in equal amounts to construct resistant and susceptible DNA pools, and sending them to the company for BSA-seq sequencing to obtain basic information for preliminary positioning. Given the large number of positioning intervals and the large range, insertion-deletion (InDel) markers were developed in the target region for verification based on the resequencing data of both parents.

[0029] (2) Extraction of parents HN1-1 and 159 and F 2The leaf genomic DNA of each plant in the population was obtained. The polymorphism of the two parents was screened by the verification markers obtained in step (1). The PCR reaction was carried out on a BIOER amplification instrument. PCR reaction system: DNA template 0.5 μL, 10 μM primer 0.3 μL, 10 mM dNTP 0.2 μL, 10× PCR Buffer (containing Mg 2+ )1μL, TaqDNA polymerase (5U / μL)0.1μL, add ddHO 2 O to make up to 10 μL. The length of the amplified product of the InDel primers used in this experiment is generally between 100-300 bp, and the PCR reaction program used is: 94℃4min, 94℃30sec, 60℃30sec, 72℃45sec, 34 cycles, 72℃10min. The different conditions used by different primers are mainly reflected in the annealing temperature, which is slightly adjusted individually.

[0030] The amplified products were separated by 3% agarose gel, and the amplified DNA bands were observed and recorded by gel imaging. 2 Amplification analysis was performed in the population to obtain F 2 Genotype information of the population.

[0031] (3) According to the linkage marker and F 2 The genotype information of the isolated population was used to construct a partial genetic map of wax gourd based on the linkage exchange law using the software JoinMap3.0 and obtain the genetic distance of each molecular marker. 2 The molecular marker genotype information of individual plants in the population and the corresponding wilt resistance value were used to scan the QTL loci of the target chromosome using the composite interval mapping method of MapQTL 5 software.

[0032] 3. Results and analysis

[0033] Donggua 159, HN1-1 and F 1 Phenotypic identification of plant resistance to Fusarium wilt Figure 1 As shown in the figure, the identification results of winter melon resistance to Fusarium wilt show that the average disease resistance level of HN1-1 is 0, while 159 is 5, and F 1 5, indicating that HN1-1 is highly resistant to Fusarium wilt, 159 and F 1 Highly susceptible to Fusarium wilt, where the resistance gene is recessive.

[0034] Winter melon 159 / HN1-1 F 2 The phenotypic distribution of the population's resistance to Fusarium wilt is shown in the figure below. Figure 2 As shown in the figure, 1082 F 2The frequency distribution of the average disease resistance value of individual plants in the population to Fusarium wilt was continuous, with a minimum value of 0 and a maximum value of 5. Combining the phenotypic results of the sixth generation of HN1-1 and 159, F 2 The coefficient of disease-resistant plants in the population (resistance value ≤ 3): the coefficient of disease-susceptible plants (resistance value ≥ 4) was 282: 800 (χ 2 =0.05<χ 2 0.01,1 =3.84), and the chi-square test showed that it conformed to the separation law of a major effect gene.

[0035] The F 2 The genotype and resistance level of each plant in the population. The location map of the major gene fob2 for resistance to wilt disease in winter melon material HN1-1 on chromosome 9 was obtained after QTL scanning. Figure 3 As shown in the figure, the vertical value on the horizontal axis represents the genetic distance (cM) between markers, and the vertical axis represents the LOD value. The results show that there is a significant QTL locus between the markers 9M42.56 and 9M46.89 on chromosome 9, with an LOD value of 57.9 and a resistance contribution rate of 95.1%. The gene was named fob2. Then, molecular markers 9M42.56 and 9M46.89 were used to screen recombinant single plants and backcross breeding positive single plants.

[0036] Therefore, the use of the above molecular markers to identify the presence of the fob2 resistance gene has a very high efficiency, which greatly improves the breeding progress of winter melon wilt-resistant lines or varieties.

[0037] The primer pairs and their sequences developed by the method of this embodiment are: the upstream primer sequence of primer pair 9M42.56 is: 5'-CCTTATTTAACCGATGTGAT-3'; the downstream primer sequence is: 5'-AGATTGGGAAGTCTTAGAGC-3' (SEQ ID NO.1-2). The upstream primer sequence of primer pair 9M46.89 is: 5'-GGATCTCCTACATGGTG CCT-3'; the downstream primer sequence is: 5'-TAATGCTTTAGGCTGAAGAG-3' (SEQ ID NO.3-4).

[0038] Example 2

[0039] This example verifies the molecular markers obtained in Example 1. The specific verification method is as follows:

[0040] 1. Materials and Methods

[0041] (1) Materials: 1 part of wilt-resistant winter melon material HN1-1, 1 part of wilt-susceptible winter melon material 159, 10 parts of 159×HN1-1 F 2:3Pedigree of the population, 4 negative wax gourd materials that do not carry the fob2 gene. Molecular marker primers: 9M42.56 and 9M46.89.

[0042] (2) Method: The genomic DNA of the leaves of the sample to be tested was extracted by CTAB extraction method, and the sample DNA was amplified using primer pair 9M42.56 and 9M46.89. The amplified products were separated by 3% agarose gel, and the amplified DNA bands were observed and recorded by gel imaging (the amplification method was the same as in Example 1).

[0043] 2. Results:

[0044] The above method was used to perform PCR amplification on the genomic DNA of 16 different samples of wax gourd materials, including HN1-1 and 159. The results are as follows: Figure 4-Figure 5 As shown. The results showed that the primers 9M42.56 (or 9M46.89) could amplify a fragment of 294 bp (or 159 bp) in the positive samples, but these fragments could not be amplified in the negative samples. This shows that the molecular marker method provided by the present invention can accurately screen out samples containing the major effect gene fob2 for resistance to wilt disease, thereby greatly improving the selection efficiency of positive individual plants of wax gourd breeding materials.

[0045] Figure 4 and Figure 5 Agarose gel electrophoresis detection of the amplified products of molecular markers 9M42.56 and 9M46.89 in different wax gourd materials, where M is BM2000 DNA marker, and the band sizes from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, respectively; R is HN1-1, S is 159, and lanes 1-10 are partial 159 / HN1-1 F 2:3 Family, lanes 11-14 are 4 negative wax gourd materials that do not carry the fob2 gene; the arrows point to the target band of the resistant parent. In the positive samples, there are target bands (294bp, 9M42.56, Figure 4 ; or 159bp, 9M46.89, Figure 5 ), while this band was not present in negative samples.

[0046] In summary, the molecular markers 9M42.56 and 9M46.89 developed in the present application are closely linked and co-segregated with the major wilt resistance gene fob2. The above molecular markers can be used to effectively distinguish whether winter melon is resistant to wilt pathogens, that is, the resistance of winter melon plants to wilt can be accurately predicted. The detection is convenient, fast and easy, and is not affected by the environment. It can quickly screen breeding intermediate materials, accelerate the breeding of disease-resistant lines or varieties, assist in clarifying breeding goals, and save costs.

[0047] Example 3

[0048] Phenotypic verification: From the electrophoresis results of Example 2, the wax gourd materials in lane R (highly resistant material HN1-1), lane S (highly susceptible material 159) and lanes 1 to 14 were selected for planting to conduct phenotypic verification of Fusarium wilt.

[0049] The specific method is as follows:

[0050] The root system bacterial solution immersion method was used to identify the resistance of wax gourd to wilt disease. The strain was the Fusarium oxysporum Schl. f. sp. Benincasae isolated, identified and preserved by the project team. The culture medium was made of corn flour: water: rice bran in a ratio of 2:5:1. After inoculation, it was cultured in an incubator at 30°C for 6 days. After the pathogen produced a large number of spores, it was prepared into a concentration of 5×10 5 / mL of spore solution of wax gourd wilt pathogen; 15 wax gourd seedlings to be verified at the 2-leaf 1-heart stage were inoculated by root immersion method, and after the roots were immersed in the solution for 5 minutes, they were transplanted to cultivation bags (h=20 cm, d=20 cm) for planting and routine production management was carried out; the disease condition of the plants was observed every day, and records were made when the highly susceptible material 159 (lane S) began to develop the disease, and records were made once every 2 days until the disease index of 159 was close to level 4, and the disease index of each material was recorded.

[0051] According to the disease value, the disease conditions of the above materials were divided into 6 resistance levels, with levels 0 and 1 representing high resistance levels, levels 2 and 3 representing intermediate types, and levels 4 and 5 representing high sensitivity levels. Level 0 means healthy plant growth without leaf damage; level 1 means one true leaf wilted; level 2 means two true leaves wilted; level 3 means three true leaves wilted; level 4 means four true leaves wilted; level 5 means all cotyledons wilted and died. The resistance level of each strain is the weighted average of the measured levels of each repeated strain.

[0052] The results are shown in Table 1:

[0053] Table 1 Disease resistance score (DRS) of materials in different lanes

[0054] Lanes DRS Lanes DRS Lanes DRS Lanes DRS R 0 4 1 9 0 14 5 S 5 5 0 10 4 1 3 6 5 11 5 2 1 7 4 12 5 3 0 8 5 13 5

[0055] As can be seen from Table 1, when the disease resistance level of lane S (highly susceptible material 159) reached level 5, the disease susceptibility levels of the plants in lanes 6-8 and 10-14 all reached levels 4-5, indicating that the plant samples in these lanes, like lane S (highly susceptible material 159), were highly susceptible plants; while the disease resistance levels of lane R and lanes 2-5 and 9 were still 0, indicating that the plant samples in lanes 10 and 13, like lane R (highly resistant material HN1-1), were highly resistant plants; and the disease resistance level of lane 1 was between 2-3, indicating that the resistance of these samples to wax gourd wilt disease was between highly susceptible and highly resistant.

[0056] Therefore, combined with Example 2, in summary, molecular markers 9M42.56 and 9M46.89 can be used to breed wax gourd strains or varieties with high resistance to Fusarium wilt disease. The specific method is as follows:

[0057] Primer pair 9M42.56 is used to amplify the genomic DNA of the wax gourd sample to be tested. If a 294 bp target fragment can be amplified, it means that the wax gourd sample contains the major gene fob2 for resistance to wax gourd wilt disease. If a 294 bp target fragment cannot be amplified, it means that the wax gourd sample does not contain the major gene fob2 for resistance to wax gourd wilt disease.

[0058] Or use primer pair 9M46.89 to amplify the genomic DNA of the wax gourd sample to be tested. If the target fragment of 159 bp can be amplified, it means that the wax gourd sample contains the major gene fob2 for resistance to wax gourd wilt disease; if the target fragment of 159 bp cannot be amplified, it means that the wax gourd sample does not contain the major gene fob2 for resistance to wax gourd wilt disease.

[0059] In summary, the main effect gene detected by the molecular marker developed in this application is clearly located and easy to identify. By detecting the molecular markers closely linked to the gene, the resistance of winter melon plants to wilt disease can be accurately distinguished, and the detection is convenient, fast and convenient, and is not affected by the environment. Positive individual plants carrying resistance genes can be identified at the seedling stage of winter melon, and other plants that do not carry resistance genes can be eliminated, which greatly improves the screening efficiency of winter melon resistance to wilt disease, saves production costs, and promotes the breeding process of disease-resistant winter melon lines or varieties.

[0060] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. The use of a primer set for detecting molecular markers of the major gene fob2 for resistance to wilt disease in wax gourd in breeding wax gourd strains or varieties with high resistance to wilt disease, characterized in that: The primer set includes primer pair 9M42.56 and / or primer pair 9M46.89; the nucleic acid sequence of the primer pair 9M42.56 is shown as SEQ ID NO.1-2; the nucleic acid sequence of the primer pair 9M46.89 is shown as SEQ ID NO.3-4; and the wax gourd material is HN1-1.

2. A method for breeding wax gourd strains or varieties with high resistance to Fusarium wilt using the primer set as claimed in claim 1, characterized in that: The method is: Primer pair 9M42.56 is used to amplify the genomic DNA of the wax gourd sample to be tested. If a 294 bp target fragment can be amplified, it means that the wax gourd sample contains the major gene fob2 for resistance to wax gourd wilt disease. If a 294 bp target fragment cannot be amplified, it means that the wax gourd sample does not contain the major gene fob2 for resistance to wax gourd wilt disease. Or use primer pair 9M46.89 to amplify the genomic DNA of the wax gourd sample to be tested. If the target fragment of 159 bp can be amplified, it means that the wax gourd sample contains the major gene fob2 for resistance to wax gourd wilt disease; if the target fragment of 159 bp cannot be amplified, it means that the wax gourd sample does not contain the major gene fob2 for resistance to wax gourd wilt disease.

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